From a Droplet to a Fibril and from a Fibril to a Droplet: Intertwined Transition Pathways in Highly Dynamic Enzyme-Modulated Peptide-Adenosine Triphosphate Systems.

Many cellular coassemblies of proteins and polynucleotides facilitate liquid-liquid phase separation (LLPS) and the subsequent self-assembly of disease-associated amyloid fibrils within the liquid droplets. Here, we explore the dynamics of coupled phase and conformational transitions of model adenosine triphosphate (ATP)-binding peptides, ACC1-13Kn, consisting of the potent amyloidogenic fragment of insulin's A-chain (ACC1-13) merged with oligolysine segments of various lengths (Kn, n = 16, 24, 40). The self-assembly of ATP-stabilized amyloid fibrils is preceded by LLPS for peptides with sufficiently long oligolysine segments. The two-component droplets and fibrils are in dynamic equilibria with free ATP and monomeric peptides, which makes them susceptible to ATP-hydrolyzing apyrase and ACC1-13Kn-digesting proteinase K. Both enzymes are capable of rapid disassembly of amyloid fibrils, producing either monomers of the peptide (apyrase) or free ATP released together with cleaved-off oligolysine segments (proteinase K). In the latter case, the enzyme-sequestered Kn segments form subsequent droplets with the co-released ATP, resulting in an unusual fibril-to-droplet transition. In support of the highly dynamic nature of the aggregate-monomer equilibria, addition of superstoichiometric amounts of free peptide to the ACC1-13Kn-ATP coaggregate causes its disassembly. Our results show that the droplet state is not merely an intermediate phase on the pathway to the amyloid aggregate but may also constitute the final phase of a complex amyloidogenic protein misfolding scenario rich in highly degraded protein fragments incompetent to transition again into fibrils.

Morphological Transformations of SARS-CoV-2 Nucleocapsid Protein Biocondensates Mediated by Antimicrobial Peptides.

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.

Clues to the Design of Aggregation-Resistant Insulin from Proline Scanning of Highly Amyloidogenic Peptides Derived from the N-Terminal Segment of the A-Chain.

Insulin aggregation poses a significant problem in pharmacology and medicine as it occurs during prolonged storage of the hormone and in vivo at insulin injection sites. We have recently shown that dominant forces driving the self-assembly of insulin fibrils are likely to arise from intermolecular interactions involving the N-terminal segment of the A-chain (ACC1-13). Here, we study how proline substitutions within the pilot GIVEQ sequence of this fragment affect its propensity to aggregate in both neutral and acidic environments. In a reasonable agreement with in silico prediction based on the Cordax algorithm, proline substitutions at positions 3, 4, and 5 turn out to be very effective in preventing aggregation according to thioflavin T-fluorescence-based kinetic assay, infrared spectroscopy, and atomic force microscopy (AFM). Since the valine and glutamate side chains within this segment are strongly involved in the interactions with the insulin receptor, we have focused on the possible implications of the Q → P substitution for insulin's stability and interactions with the receptor. To this end, comparative molecular dynamics (MD) simulations of the Q5P mutant and wild-type insulin were carried out for both free and receptor-bound (site 1) monomers. The results point to a mild destabilization of the mutant vis à vis the wild-type monomer, as well as partial preservation of key contacts in the complex between Q5P insulin and the receptor. We discuss the implications of these findings in the context of the design of aggregation-resistant insulin analogues retaining hormonal activity.